Human Artificial Chromosomes (HACs) assembled from alphoid DNA arrays represent novel vectors that have a great potential for the study of assembly and maintenance of the human kinetochore as well as for gene therapy, for screening of anticancer drugs and for biotechnology. We previously constructed a synthetic HAC (tetO-HAC) allowing tethering of its kinetochore by different chromatin modifies fused with the tet-repressor protein. This HAC was successfully used to clarify a role of different types of chromatin in function of the kinetochore. During the past year, the same approach exploiting the tethering of the tetO-HAC kinetochore revealed that the kinetochore protein CENP-C coordinates assembly and function of the outer kinetochore with the epigenetic maintenance of CENP-A chromatin. In another study, we demonstrated that KAT7-containing acethyltransferases associated with the Mis18 complex provides competence for histone turnover/exchanges activity on centromeric DNA repeats and prevents Suv39h1-mediated heterochromatin invasion into centromeres. We have also used our tetO-HAC for development of new assays for measuring chromosome instability (CIN) in human cells. Whole-chromosomal instability (CIN), manifested as unequal chromosome distribution during cell division, is a characteristic feature of most types of cancer, thus distinguishing them from their normal counterparts. Although CIN is generally considered as a driver of tumor growth, a threshold level exists whereby further increase in CIN frequency becomes a barrier against tumor growth and, therefore, can be exploited therapeutically. However, drugs known to increase CIN beyond this therapeutic threshold are currently few in number. In our previous work, we have described a quantitative assay for measuring CIN based on the use of a non-essential HAC carrying a constitutively expressed EGFP transgene (this assay was named as loss of signal assay). Using this assay, more than 100 anticancer drugs were ranked on their effect on HAC loss. However, it was problematic to convert loss of signal assay into high-throughput screen of chemical libraries and for identification of new CIN genes that are required for proper chromosome transmission. To address this point, we re-designed the HAC-based quantitative assay. In a new assay, the HAC carries a constitutively expressed shRNA against the EGFP transgene integrated into human genome. Thus, cells that inherit the HAC display no green fluorescence, while cells lacking the HAC do. We verified accuracy of this gain of signal assay by measuring the level of CIN induced by known antimitotic drugs and added to a list of the previously identified compounds exhibiting the highest effect on HAC loss a newly characterized inhibitor of the centromere-associated protein CENP-E, PF-2771. The gain of signal assay was also sensitive enough to detect increase of CIN after siRNA depletion of genes controlling mitotic progression through distinct mechanisms, indicating its application for screening unknown yet genes controlling chromosome transmission. A work with a new system for high-throughput screening using a fluorescence microplate reader to characterize chemical libraries and identification of new conditions that modulate CIN levels is in progress. We previously demonstrated the utility of tetO-HAC-based vectors for gene delivery and complementation of gene deficiencies in human cells. The tetO-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of its kinetochore via binding of chromatin modifiers, such as tTS or tTA, to its centromeric tetO sequences. The opportunity to induce HAC loss provides a unique control for phenotypes induced by genes loaded into the tetO-HAC. A wide use of HAC vectors is limited significantly by the difficulty of their transfer from one type of cells to other cells. So far, HAC transfer is performed via micro-cell mediated chromosome transfer (MMCT) procedure developed for transfer of entire chromosomes. For the past decades, the MMCT technique has been applied for transferring HACs carrying entire chromosomal copies of genes for genes function studies. However, a safe and highly efficient MMCT technique remained an important challenge. In our recent work, we modified the MMCT method and demonstrated that the change of Colcemid plus Cytochalasim B for TN16+Griseofulvin plus Latranculin B in combination with plastic modification (Collage/Laminin) greatly increases the efficiency of HAC transfer to recipient cells and in addition made the HAC transfer safer. The improved MMCT protocol was successfully applied for HAC transfer into several different recipient cells lines, including human stem cells.